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. 2009 Apr;20(2):116-27.

Identification of optimal protocols for sequencing difficult templates: results of the 2008 ABRF DNA Sequencing Research Group difficult template study 2008

Jan Kieleczawa  1 , Debbie Adam, Doug Bintzler, Michelle Detwiler, David Needleman, Peter Schweitzer, Sushmita Singh, Robert Steen, Michael Zianni
Affiliations

Identification of optimal protocols for sequencing difficult templates: results of the 2008 ABRF DNA Sequencing Research Group difficult template study 2008

Jan Kieleczawa et al. J Biomol Tech. 2009 Apr.

Abstract

The 2008 ABRF DNA Sequencing Research Group (DSRG) difficult template sequencing study was designed to identify a general set of guidelines that would constitute the best approaches for sequencing difficult templates. This was a continuation of previous DSRG difficult template studies performed in 1996, 1997, and 2003. The distinguishing factors in the present study were the number of DNA templates used, the number of different types of difficult regions tested, and the inclusion of a follow-up phase of the study to identify optimal protocols for each type of difficult template. DNA templates with associated sequencing primers were distributed to participating laboratories and each laboratory returned their sequencing results along with descriptions of the experimental conditions used. The data were analyzed and the best protocols were identified for each difficult template. This information was subsequently distributed to the participating laboratories for a second round of sequencing to evaluate the general applicability of the optimized protocols. The average improvements in sequencing results were 11% overall, with a range of -25% to +43% using the optimized protocols. The full results from this study are presented here and they demonstrate that general experimental protocols and common additives can be used to improve the sequencing success for many difficult templates.

Keywords: DNA sequencing; difficult template; research group study.

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Figures

FIGURE 1
FIGURE 1
Assessing the quantity and integrity of difficult templates used in the DSRG study. Aliquots of about 200 ng of each DNA were run on a 1% agarose gel using standard molecular biology protocols. Lanes 1–8 indicate DNAs 1–8. Lane 9 is the low DNA mass ladder and lane 10 is the supercoiled DNA ladder. The top band in lane 9 contains 200 ng of DNA, and by comparison, all 8 DSRG difficult templates have very similar amounts of DNA.
FIGURE 2
FIGURE 2
The average Q ≥20 scores for each template/primer combination from laboratories returning data for both phase I and phase II. The average Q ≥20 scores for each template/primer combination from Table 4 are shown graphically. The overall average increase in Q ≥20 scores was +11%, with a range of −25% to +43%.
FIGURE 3
FIGURE 3
The average (A) and the most optimal (B) results for DNA 1. Note that for an average protocol, the sequence stops after approximately 300 bases, whereas the most optimal protocol yields sequence data passed 900 bases. Horizontal red bars indicate the same region in each chromatogram. In C, the various potential difficult motifs in this template are shown graphically. Nonrepeat (DN) is the long (default in Examine Repeat module of 4D LIMS is 40 bases) stretch of two bases that do not form dinucleotide repeats. Type, Length, Count, and Positions indicate the kind, length, number of occurrences, and positions in the template, respectively.
FIGURE 3
FIGURE 3
The average (A) and the most optimal (B) results for DNA 1. Note that for an average protocol, the sequence stops after approximately 300 bases, whereas the most optimal protocol yields sequence data passed 900 bases. Horizontal red bars indicate the same region in each chromatogram. In C, the various potential difficult motifs in this template are shown graphically. Nonrepeat (DN) is the long (default in Examine Repeat module of 4D LIMS is 40 bases) stretch of two bases that do not form dinucleotide repeats. Type, Length, Count, and Positions indicate the kind, length, number of occurrences, and positions in the template, respectively.
FIGURE 4
FIGURE 4
The average (A) and the most optimal (B) results for DNA 3. Note that for an average protocol, the sequence deteriorates after approximately 200 bases. The most optimal protocol yields sequence data past 800 bases, although significant background noise is observed. Horizontal red bars indicate the same region in each chromatogram. In C, the various potential difficult motifs in this template are listed as in Figure 3.
FIGURE 4
FIGURE 4
The average (A) and the most optimal (B) results for DNA 3. Note that for an average protocol, the sequence deteriorates after approximately 200 bases. The most optimal protocol yields sequence data past 800 bases, although significant background noise is observed. Horizontal red bars indicate the same region in each chromatogram. In C, the various potential difficult motifs in this template are listed as in Figure 3.
FIGURE 5
FIGURE 5
The average (A) and the most optimal (B) results for DNA 5. Note that for an average protocol, the sequence deteriorates after approximately 300 bases, and the most optimal protocol yields sequence data to approximately 600 bases. Horizontal red bars indicate the same region in each chromatogram. In C, the various potential difficult motifs in this template are listed as in Figure 3 (only 130 bases in this nonrepeat are shown here).
FIGURE 5
FIGURE 5
The average (A) and the most optimal (B) results for DNA 5. Note that for an average protocol, the sequence deteriorates after approximately 300 bases, and the most optimal protocol yields sequence data to approximately 600 bases. Horizontal red bars indicate the same region in each chromatogram. In C, the various potential difficult motifs in this template are listed as in Figure 3 (only 130 bases in this nonrepeat are shown here).
FIGURE 6
FIGURE 6
The average (A) and the most optimal (B) results for DNA 8. Note that for an average protocol, the sequence deteriorates after approximately 600 bases. The most optimal protocol yields sequence data over 1000 bases, although significant background noise is observed. Horizontal red bars indicate the same region in each chromatogram. In C, the various potential difficult motifs in this template are listed as in Figure 3.
FIGURE 6
FIGURE 6
The average (A) and the most optimal (B) results for DNA 8. Note that for an average protocol, the sequence deteriorates after approximately 600 bases. The most optimal protocol yields sequence data over 1000 bases, although significant background noise is observed. Horizontal red bars indicate the same region in each chromatogram. In C, the various potential difficult motifs in this template are listed as in Figure 3.
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